Propionic acid fermentation process



Patented Nov. s, 1935 UNITED STATES PATENT OFFICE PROPIONIC ACID FERMENTATION PROCESS Hugh. It. Stiles, Terre Hautc, Ind., assignor to Commercial Solvents Corporation, Terre Haute, Ind., a corporation of Maryland No Drawing. Application June 29, 1932, Serial No. 619,965

16 Claims. (Cl. 260-120) 1 fermentation has, in most cases, been too slow for practical purposes. 1,459,959 of Sherman et al., discloses a method for accelerating this fermentation by means of lactic acid organisms and certain other bacteria. However, even with this improvement, the fermentation has been undesirably slow and the yields have not been greatly improved.

While catalytic agents are well known in chemistry, their use in the fermentation field has been practically negligible. The catalytic effect of symbiotic action of bacteria hasbeen utilized, and

minute quantities of certain soluble substances cal catalysis, havenot thus far been utilized in the fermentation field. Certain solid supporting mediahave been used, for example, the wood shavings employed in the quick vinegar process. However, these materials merely serve to retain the bacteria and do not have any specific catalytic effect upon the fermentation.

The surprising discovery has now been made that a definite catalytic eifectupon the propionic acid fermentation is secured by including in the media certain solid contact agents. The agents which have been found to be suitable for this purpose are the clays known as bentonites and the hydrous aluminium oxides. Of the latter compounds, it is preferred to use the partially dehydrated products known as activated alumina. Particularly adapted to the present process are 2. grade of bentonite known as bentonite B and an activated alumina known as 1228 G.

. Bentinite B is a grade of lay having the following approximate composition:

Per cent MgO 2.6

NazO u 2.3 Ignition loss- Y United States Patent No,

drawn at each draw-ofi when activated alumina Activated alumina 1228 G is a partially dehydrated alumina trihydrate which has an approximate specific gravity of 3.25, is granular in form, and very resistant to crushing and abrasion.

Its approximate analysis is as follows: 5

- Per cent S102 0.03 A1201 91.23 F620 0.01 TiOa 0.01 10 NazO 1.27 Ignition loss 7.10

' mash since there will be less absorbed upon the solid material. Likewise, if it should be desired to operate the fermentation in a semi-continuous manner more of the fermented liquid can be with- 25 is used as the catalyst.

The use of these materials is adapted to fermentations by means of any of the known types. of propionic acidbacteria, for example those described in The Propionic Acid Bacteria, C. B. Van Niel; N. V. Uitgeverszaak J. W. Boissevain & C0. Haarlem, 1928. Especially suited for this purpose is bacterium acidi propionici, an organism isolated from Swiss cheese; and various strains 5 of this organism may be employed, as'for example, bacterium acidi propionici, bacterium acidi propionici strain a, strain b, strain 0, strain d, or strain rubrum". I

In addition to theproponic acid bacteria, there 40 may be employed known accelerating organisms such as those disclosed in United States Patent No. 1,459,959 of Sherman et a1. For example there may be used: 1. Those organisms which produce lactic acid from carbohydrates, as for 5 example, those described on pages 241-255 of Bergeys Manual of Determinative Bacteriology (1923 edition); 2. Organisms which hydrolyze nitrogenous substances and form therefrom new materials which can be acted upon by other bac- 50 fall into either of the preceding groups but which ferment salts of organic acids such as the mem- 55 bers of the Alcaligines group (pages 233-237 of Bergeys Manual of Determinatlve Bacteriology).

The present process is particularly adapted to be employed in conjunction with the process described in co-pending patent application Serial No. 619,964 flled June 29, 1932 by H. R. Stiles, now Patent No. 1,946,447, dated Feb. 6, 1934. According to the method of this application, fructose-containing mashes are subjected to the action of mannitol-forming bacteria and lactic acid bacteria, and the resulting, at least partially fermented, mash is subjected to the action of proponic acid bacteria. The mannitol-forming bacteria for this process may be of any of the known types, such as for example, bacterium mannitopoeum, bacillus gayoni, and lactobacillus .fermentum, or any of the organisms described in "Fermentation Products of Certain Mannitol- Forming Bacteria, H. R. Stiles et al., Journal of Biological Chemistry 64, 643, 1925. The lactic acid bacteria may also be any of the known types such as those included in group 1 above. This fermentation may be carried out by either the simultaneous or successive inoculation with the mannitol and lactic organisms and the propionic acid organisms. Thus, the fermentation with the mannitol and lactic organisms may be allowed to go to completion before inoculating with the propionic acid bacteria, or the fermentation may be accomplished by the simultaneous action of all three types of bacteria. The present process is particularly applicable to the latter method of fermentation.

Aside from the presence of the catalytic agents referred to, the mashes used in the present process may be of any type known to be suitable for the particular type of propionic acid fermentation employed. Thus, if the ordinary propionic acid fermentation or the process of United States Patent No. 1,459,959 referred to above, are utilized, practically any soluble carbohydrate may be employed, such as for example, glucose, mal- .tose, lactose, hydrol (the molasses obtained in the corn sugar industry), hydrolyzed grain, and the like. If the process of co-pending application Serial No. 619,964 is employed, mashes should be used which contain soluble carbohydrate of which 25% is fructose or its equivalent in the form of fructose-yielding carbohydrates such as sucrose, raflinose, gentianose, inversion products of these sugars such as the commonly known invert sugar from sucrose, molasses, inverted molasses, hydrolyzed inulin, Jerusalem artichoke tubers, and the like.v

Known nutrients such as soluble nitrogen in the form of yeast water, steep water, butyl-acetonic slop or distillery wastes from other fermentation industries may be employed. The hydrogen ion concentration may be maintained within the same limits as suitable for the fermentation by propionic acid bacteria alone, e. g., a pH of 5.4-6.0, the pH maintained by an excess of calcium carbonate in the medium. The fermenta'tion temperature may likewise be maintained within the limits known to be operative for the propionic fermentation. The preferred temperature, however, is 30-31 C. In all other respects, such as the mechanical steps in carrying out the fermentation, the known methods for the propicnic acid fermentation may be employed.

In any of the above type of mashes, the agents may be employed in concentrations of from 0.5 to 5% by weight of the mash, but are preferably used in concentrations of from 1 to 3%. This material may, of course, be added to the sterilized mash in a sterile condition, but it is more conveniently added to the mash before sterilization in the ordinary manner.

The present invention may perhaps best be illustrated by the following specific examples: 5

Example I A mash containing 7% of hydrolyzed corn, (i. e., approximately 5% total sugar), 0.6% of steep water (dry basis), 3% of calcite and 3% of acti- 10 vated alumina is inoculated with approximately- 5% of a culture containing propionic acid bacteria and10'% lactic acid bacteria and the fermentation is allowed to proceed at 30 C. in the usual manner for the propionic acid fermen-'15 tation.

Example II A mash containing 10% of molasses (i. e., approximately 5% total sugar), 0.6% of steep water (dry basis), 3% of calcite and 3% of bentonite B is inoculated with about 2% by volume of a culture containing approximately 90% propionic acid bacteria, 5% mannitol-forming bacteria, and 5% lactic acid bacteria. The fermentation is allowed to proceed at 30 C. in the usual mane ner for the propionic acid fermentation.

Example. III

A' mash of inverted molasses diluted with butyl-acetonic slop to a concentration of about 30 10%, (i. e. approximately 5% of invert sugar), containing 3% of calcite and 3% of activated alumina is inoculated with 2% of a culture containing approximately 50% of mannitol-forming 35 bacteria and approximately 50% of lactic acid bacteria. The fermentation is allowed to go to completion and the resulting mash is then inoculated with propionic acid bacteria. The fermentation is then allowed to continue in the usual manner for the propionic acid fermenta- 40 tion.

The fermentation may of course, if desired, be carried out in a continuous or semi-continuous manner, as well as by the usual batch process 5 of the above examples. The following example illustrates a method for carrying out the fermentation in a semi-continuous manner:

Example IV A mash containing approximately 10% of in- 50 verted molasses. (i. e. approximately 5% invert sugar), 0.6% of steep water (dry basis), 3% of calcite and 3% of bentonite.B is inoculated with a mixed culture containing approximately 90% 55 of propionic acid bacteria in the form of a culture containing bacterium acidi propionici, bacterium acidi propionici strain 1), strain 0, or strain rubrum", approximately 5% of lactobacillus casei and approximately 5% of lactobacil- 60 lus fermentum. The fermentation is allowed to proceed at 30 C. for seven days at which time the fermented mash is allowed to settle,'50% of the mash is withdrawn, preferably from the top of the vessel, and an equal portion of fresh 65 mash is introduced. In this manner a semi-continuous fermentation may be carried out indefinitely so long as contamination or other difficulties are not encountered.

The results in the following table show the 70 beneficial results secured by the use of these catalytic agents, Other solid contact materials were employed in fermentations carried out in parallel with the alumina and bentonite fermentation. One fermentation contained no con- 75 2,020,201 tact agent and the others contained one and 3% respectively, of activated alumina, bentonite B and silica gel. The medium employed contained 10% of molasses, approximately 0.6% butylacetonic slop (dry basis) and 5% of calcite. A

mixed culture of propionic acid bacteria, mannitol-iormlng organisms, and lactic acid bacteria was used. The media were sterilized for one hour at 20 lbs. pressure. The Iermentations were carried out' at 30 C., were thoroughly agitated once each day, and yields were determined at 9, 12 and 15 days.

proved results secured by the use of the catalytic agents and shows the particular advantage secured by the use-of these agents in the mannitol type fermentation. The fermentation medium TABLE I Volatile Propionlc Lactic Contact agent acid acid acid Sugar leit Alcohol Result: alter 9 days Percent 6/100 o. a} total 6/100 cc. 6/100 cc. None. 1 48 56 2. 56 0. 33 1% Act. alumina 2. 36 03 1. 57 0.31 1% Bentonite B. l. 84 58 l. 97 0. 29 1% Silica el l. 45 56 2. 53 0. 31 3% Act. alumina 3. 22 70 l. 20 0. 34 3% Bentonite B 2. 37 04 1. 57 0. 32 3% Silica gel 2. 15 61 1. 69 0. 32

Results ajter 12 days None 2. 18 63 1.88 0. 28 1% Act. alumina Q 3. 12 61 0. 93 0. 28 1% Bentonite 13.- 2. 58 67 l. 40 0.25 1% Silica gel..- 2. 12 64 1. 84 3% Act. alumina 3. 22 68 0.88 0.25 3% Bentonito B 2. 92 67 1. 04 0. 31 3% Silica m 2. 22 67 1. 74 0. 23

- Results ajter 16 days None. 2. 83 67 0. 32 6. 3 1% Act. alumina 3. 47 59 0. 57 0. 31 5. 9 1% Bentonite B- 3. 22 67 0.92 0. 29 5. 9 1% Silica el 2. so 1. 2-1 0. 33 5. 6 3% Act. l min i 3. 55 69 0.61 0.32 5. 0 3% Bentonite B 3. 41 70 0. 78 0. 33 4. 8 3% Silica o] 2. 94 68 l. 07 0. 32 5. 0

The following experiment, carried out in a employed was the same as in the above experisemi-continuous manner, also illustrates the 1mment and the same conditions were utilized with the exception that at the end of every seven days, 50% of the fermented mash was withdrawn and was replaced by an equal volume of fresh mash. The results are given below.

TABLE II Volatile Propionic Lactic Sugar 7 e Contact agent acid acid acid Mt Alcohol Results 0] lat draw of 1 Percent 6/100 cc. of total 6/100 cc. 6/100 cc. G/lller 3% Silica gel 1. 25 49 3. l0 0. 38 6. 9 3% Act. alumina l. 56 57 2. 39 0. 21 5.9 3% Kieselguhr 1. 33 51 2. 72 0. 26 7. 1 3% Bentonite R l. 46 55 3. 02 0. 26 8. 0

Results 012ml draw of 3% sniciiil 2 40 2.01 o. 34 6.6 3% Act. nlllmiflfi 3 3. 36 67 1. 04 0. 28 5. 2 3% Kieselguhr- 2. 57 62 1. 67 0. 29 6. 4 3% Bentonite R 2. 90 66 1.51 0. 31 5. 6

Results 01341 draw of 3% Silica g 2. 59 64 1. 47 0. 32 4. 5 3% Act. nlnmiml 3. 50 69 0. 0. 25 3. 3 3% Kieselguhr. 3.09 70 0.93 0. 31 3. 6 3% Bentonite B 3. 25 70 0. 79 0. 29 3. 4

As may be noted from the above table, when the catalytic agents of the present invention were employed, not only were the yields improved but also the ratio of propionic acid was increased and the amount of undesired alcohol was decreased.

In this type of fermentation, in the presence of both fructose and glucose types of carbohydrates, the lactic acid bacteria apparently preferentially attack the glucose and the mannitol bacteria preferentially attack the fructose. This is illustrated proximate composition:

by the improved results obtained according to the process of co-pending application Ser. No. 619,964 in which these organisms are employed without the use of catalytic agents. However, as may be seen from the above table, the catalytic agents apparently exert a directing influence upon this fermentation and secure even a further preferential attack upon the carbohydrates, thus securing not only the improved yields obtained in the ordinary propionic fermentation, but also the added advantage of a higher ratio of propionic acid in the products.

It is to be distinctly understood that the above examples are merely illustrative of the present invention and that the invention is not to be taken as limited to the particular processes described. Neither is the invention to be limited to the particular strains of bacteria or the particular types of raw materials specified. Equivalents which would naturally occur to one skilled in the art may be employed without departing from the spirit of the invention.

The invention now having been described, what is claimed is:

1. In a process for the production of propionic acid by fermentation, the step which comprises carrying out said fermentation in the presence of a catalytic agent chosen from the group consisting of bentonite clays and hydrous aluminium oxides. a

2. In a process for the production of propionic acid by fermentation of a mash containing soluble carbohydrate material of at least 25% fructose equivalent by means of the simultaneous action of mannitol-forming bacteria, lactic acid bacteria and propionic acid bacteria, the step which comprises carrying out said fermentation in the presence of a catalytic agent chosen from the group consisting of bentonite clays and hydrous aluminium oxides. v

3. In a process for the production of propionic acid by fermentation, the step which comprises carrying out said fermentation in the presence of activated alumina comprising essentially partially I dehydrated aluminium trihydrate.

Percent SiQ-z 58.0 A120 19.9 F6201 2.9 MgO 2.6 N820 .I 2.3 Ignition loss 8.1

5. In aprocess for the production of propionic acid by fermentation of a mash containing soluble carbohydrate material of at least 25% fructose equivalent by means of the simultaneous action of mannitol-forming bacteria, lactic acid bacteria and propionic acid bacteria, the step'which comprises carrying out said fermentation in the presence of bentonite clay of the following approxi- 6. In a process for the production of propionic acid by fermentation of a mesh containing soluble carbohydrate material of at least 25% fructose equivalent by means of the simultaneous action of mannitol-forming bacteria, lactic acid bacteria and propionic acid bacteria, the step which .com-

prises carrying out said fermentation in the pres- 5 ence of 1 to 3% of bentonite clay of the following approximate combination.

Ignition loss 8.1

7. In a process for the production of propionic l5 acid by fermentation, the step which comprises carrying out said fermentation in the presence of a partially dehydrated hydrous aluminium oxide.

8. In a process for the production of propionic 20 acid by fermentation, the step which comprises carrying out said fermentation in the presence of 1 to 3% of a partially dehydrated hydrous aluminium oxide.

9. In a process for the production of propionic 25 acid by fermentation of a mash containing soluble carbohydrate material of at least 25% fructose equivalent by means of the simultaneous ac tion of mannitol-forming bacteria, lactic acid bacteria, and propionic acid bacteria, the step 30 which comprises carrying out said fermentation in the presence of 1 to 3% of a partially dehydratedhydrous aluminium oxide.

10. In a process for the production of propionic acidby fermentation, the step which comprises carrying out said fermentation in the presence of a bentonite clay having an A1203 content of about20%.

11. In a-process for the production of propionic acid by fermentation, the step which comprises carrying out said fermentation in the presence of 1 to 3% of bentonite clay.

12. In a process for the production of propionic acid by fermentation of a mash containing soluble carbohydrate material of at least 25% fructose equivalent by means of the simultaneous action of mannitol-forming bacteria, lactic acid bacteria, and propionic acid bacteria, the step whichcomprises carrying out said fermentation in the presence of 1 to 3% of bentonite clay. 13. In a process for the production of propionic acid by fermentation, the step which comprises carrying out said fermentation in the presence of activated alumina of the following approximate composition 65 g Per cent SiO2 0.08

A1203" 91.23 F8203 0.01 TiOz 0.01

NazO 1.27

-14. In a process for the production of propionic acid by fermentation, the step which comprises carrying out said fermentation in the presence of 1 to 3% of activated alumina of the following following approximate composition:

Ignition loss Per cent SiO-z 0.08 A120: 91.23

F8203 0.01 TiOz 0.01

NazO 1.27 Ignition loss 7.10

Per cent SiOz 0.08' A1203 91.23

' F8203 0.01 TiOz 0.01- NazO f 1.27 Ignition loss 7.10

15. In a process for the production of propionic acid by fermentation of a mash containing soluble carbohydrate material of at least 25% fructose equivalent by means of the simultaneous action of mannitol-forming bacteria, lactic acid bacteria, and propionic acid bacteria, the step which comprises carrying out said fermentation in the presence of activated alumina of the following approximate composition:

16. In a process for the production of propionic acid by fermentation of a mash containing solu ble carbohydrate material of at least'25%' fructose equivalent by means of the simultaneous action of mannitol-forming bacteria, lactic acid bacteria, and propionic acid bacteria, the step which comprises carrying out said fermentation in the presence of 1 to 3% of activated alumina. of the following approximate composition:

. a Per cent sioi 0.0a A1203 -Q. 91.23 F6203 0.01 TiOz u 0.01 NazO 1.27 15 Ignition loss 7.10

HUGH R. STILES. 

